ABSTRACT Addiction develops when recreational drug use switches to compulsive drug taking. While the former is predominantly motivated by reward seeking, the latter is also driven by other factors that include enhanced stress reactivity, aversive aspects of drug withdrawal and emergence of a negative affect upon protracted abstinence. In recent years the notion that reward and aversion processing engage overlapping brain circuits has been established, together with the concept of a reward/aversion network. The habenula (Hb) encodes both rewarding and aversive aspects of external stimuli, and may therefore represent a central integrator of reward/aversion circuits. Remarkably, the medial subdivision of habenula (MHb) shows highest density of mu opioid receptors (MORs) in the brain, but the role of this particular receptor population is unknown. This project will test the hypothesis that MORs expressed in the MHb regulate specific aspects of reward and aversion processes related to drug abuse. We will capitalize on tools and preliminary findings from the previous funding period. In Aim 1, we will extensively characterize MOR-expressing neurons in the medial septum-MHb-interpeduncular nucleus (MS-MHb-IPN) pathway using viral tracers combined with knock-in MOR-mcherry, Cnrb4-Cre or novel MOR-Cre mice that we currently develop. This Aim will provide circuit-level understanding that will complement Aims 2 and 3. Aim 2 will identify behaviors, and underlying circuit mechanisms, controlled by MORs in the MHb. We will examine a range of reward/aversion behaviors potentially mediated at the Hb level (reward and reward-driven decision-making, morphine and nicotine withdrawal, aversion to morphine withdrawal and abstinence) using a novel conditional Cnrb4-MOR mouse line. Reduced physical morphine withdrawal has already been detected and DREADD approaches will be used to recapitulate this behavior, and possibly other phenotypes. Aim 3 will identify the causal impact of MOR and MOR-positive neuron activities in MS-MHB-IPN networks, and their broader impact on the brain. We will use pioneering functional magnetic resonance imaging (fMRI) in live mice, and further fMRI strategies developed in the Technical Advancement Core, to map brain-wide functional connectivity, seed-based connectional patterns and inter-node directionality in Cnrb4-MOR mice at rest and after morphine treatment. Consequences of DREADD-mediated stimulation of MOR+ neurons in the MHb will also be examined by fMRI in live animals. In sum, this proposal will reveal the role(s) of the densest and less-well studied MOR population. The three aims together will determine importance of these receptors in reward/aversion-related behaviors and elucidate the underlying circuit mechanisms. The project will also provide novel genetic mouse lines for CSORDA (Cnrb4-MOR, Project III) and the neuroscience community (MOR-Cre), and cutting edge non- invasive animal imaging that will be applicable within (PTSD model, Project IV) and outside of CSORDA.